This proposal presents a program of mechanistic and spectroscopic studies of two heme-iron containing mono-oxygenases, cytochrome P-450 and secondary amine mono-oxgenase. The objectives of these experiments are to test current hypotheses about the mechanism of action of P-450, the more extensively studied of these two enzymes, and to generate mechanistic and structural ideas about secondary amine mono-oxygenase. For P-450, particular emphasis will be placed on the substrate binding process and on a re-determination of the stereochemistry of hydroxylation. In addition to experiments designed to determine the isotope effect of hydroxylation by secondary amine mono-oxygenase, spectroscopic experiments will be carried out to further characterize the oxidized and reduced enzyme in the presence and absence of substrate molecules, inhibitors and added ligands, especially dioxygen. Emphasis will be placed on the use of magnetic circular dichroism and electron paramagnetic resonance spectroscopy to provide complimentary information about these states of enzyme. This work will principally involve two methods of investigation: spectroscopic studies of stable or semi-stable states and mechanistic experiments on the actively functioning enzymes. The mechanistic studies will involve the use of rapid kinetics techniques and also the use of specially synthesized substrates.